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a Dep. of Agronomy, Purdue Univ., West Lafayette, IN 47907-1150
b Dep. of Agronomy and Range Science, Univ. of California, Davis, CA 95616-8515. Contribution from the Purdue Univ. Agric. Exp. Stn., Journal Series no. 16320
* Corresponding author (jvolenec{at}purdue.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: LSD, least significant difference • PMSF, phenyl methyl sulfonyl fluoride • SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis • TNC, total nonstructural carbohydrate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The major constraint preventing widespread use of nondormant alfalfas in temperate regions is their poor winter hardiness. Sheaffer et al. (1992) examined in spring-planted alfalfa the interactions between seeding-year harvest management, fall dormancy, and winter survival in Minnesota. They reported that fall dormancy influenced alfalfa winter hardiness more than any other feature they measured. Nondormant alfalfa cultivars died during winter irrespective of harvest management or location, whereas fall-dormant cultivars had good winter survival. These findings are supported by other studies (Smith, 1961; Barnes et al., 1979; Stout, 1985; Stout and Hall, 1989), and have resulted in fall dormancy routinely being used to predict alfalfa winter hardiness.
The mechanisms controlling the close positive association between fall dormancy and winter hardiness are not clearly understood. Genetic differences in alfalfa winter survival have been associated with several physiological changes in overwintering organs. Accumulation of starch and sugar in taproots has been positively associated with alfalfa winter survival (Graber et al., 1927; Grandfield, 1943; Smith, 1964). Accumulation of soluble sugars in roots is thought to enhance tolerance to low temperatures and other stresses associated with winter (Bula et al., 1956; Ruelke and Smith, 1956). Castonguay et al. (1995) and Castonguay and Nadeau (1998) recently reported that the accumulation of raffinose and stachyose was more closely associated with winter survival than was accumulation of starch or sucrose. Other studies indicate that N-containing compounds also accumulate in roots during winter hardening, and serve as a source of N when shoot growth is initiated in spring and for regrowing shoots after harvest (Volenec et al., 1991, 1996; Hendershot and Volenec, 1993; Avice et al., 1996). Differential gene expression and accumulation of their gene products occur during cold acclimation, and these changes also have been positively associated with genetic differences in winter survival. Changes in gene expression have been detected using in vivo or in vitro labeling of proteins (Mohapatra et al., 1987; Castonguay et al., 1993) or cDNA cloning (Laberge et al., 1993; Monroy et al., 1993; Castonguay et al., 1994).
Most studies aimed at understanding the physiological and biochemical bases for genetic variation in the relationship of fall dormancy and winter hardiness relationships used cultivars that differed in many attributes, including fall dormancy and winter survival. This large difference in genetic background makes it difficult to establish causal relationships between fall dormancy and winter hardiness. Recently we evaluated fall dormancy and winter survival of individuals from the third cycle of selection for contrasting fall dormancy created from parental lines having large initial differences in fall dormancy (Cunningham et al., 1998). Three cycles of selection for greater fall dormancy improved winter survival of CUF 101 from 1 to more than 90%. Roots and crown buds of the fall dormant, winter hardy population of CUF 101 (CUF 101-L) contained higher concentrations of sugars and proteins when compared to the nonhardy parent CUF 101.
In this study we extend our earlier work by examining plants from each of the three cycles of selection for contrasting fall dormancy in two cultivars, CUF 101 and Mesilla. We expected incremental changes in winter survival resulting from each cycle of selection for contrasting fall dormancy to parallel incremental changes in physiological and biochemical attributes important for winter hardiness. Key physiological attributes in roots that are consistently associated with selection for fall dormancy and improved winter survival can be discerned. Our objective was to determine how incremental changes in fall dormancy due to genetic selection influenced autumn height and winter survival, root physiology, and expression of a cold acclimation responsive gene family.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Seedlings were established at the Agronomy Research Center, Purdue University, West Lafayette, IN, in early May of 1997 and 1998. Seeds were sown in 3-m-long rows spaced 92 cm apart in a randomized complete-block with four replicates. Resultant plant populations were approximately 20 plants/m of linear row. The soil was a silt loam of the Starks-Fincastle series that was fertilized and limed according to soil test for high alfalfa yield. Seeds were inoculated with Rhizobium meliloti (Liphatech Corp., Milwaukee, WI) prior to planting. Plots were hand-weeded, and insects controlled as needed. Planting and sampling dates are summarized in Table 1. Plant height was measured at eight randomly selected positions within each row, and the average used as an indication of fall dormancy for that plot. Plant survival was determined when shoot growth of winter hardy cultivars resumed in April by excavating 15 to 30 plants and scoring the winter injury of each plant using the following scale: 1 = uninjured; 2 = injured; and 3 = completely dead. A weighted average was calculated for each plot and used for statistical analysis. Air and soil temperatures were recorded daily each year using National Weather Service Standard maximum and minimum thermometers (for air temperature) and a mercury thermometer for soil temperature at a 10-cm depth under bare soil (Fig. 1).
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Sugar, Starch, and Protein Analyses
Details of these analyses have been recently published (Cunningham et al., 1998). Sugars were extracted with 800 mL/L ethanol, microfuged, and the sugar concentration of the supernatant determined with anthrone (Van Handel, 1968) using glucose as a standard. Starch in the ethanol-extracted residue was gelatinized, and starch digested by adding 0.2 U of amyloglucosidase (Sigma Chemical Co., St. Louis, MO; product A3514 from Aspergillus niger) and 40 U of 
-amylase (Sigma Chemical Co., St. Louis, MO; product A0273 from A. oryzae). Tubes were centrifuged and glucose in the supernatant was determined using glucose oxidase (Glucose Trinder, Sigma Chemical Co., St. Louis, MO; product 315-100). Starch concentration was estimated as 0.9 x glucose concentration. Protein extraction was conducted at 4°C. Proteins were extracted from parallel samples using 100 mM imidazole-HCl buffer (pH 6.5) containing 10 mM 2-mercaptoethanol and 1 mM PMSF. Suspensions were centrifuged, and soluble protein in the supernatant was estimated using a protein dye-binding technique (Bradford, 1976).
RNA Isolation and Northern Blot Hybridization Analysis
Tissues were ground in liquid N2 using a mortar and pestle, and total RNA isolated using hot phenol (Ougham and Davis, 1990) with minor modifications (Gana et al., 1998). Total RNA (20 mg) was separated on a 1.5% (w/v) agarose-formaldehyde gel (Lehrach et al., 1997). The RNA was transferred by capillary action to a Zeta-probe membrane (BioRad) after electrophoresis. The membranes were pre-hybridized for 4 h at 42°C (Stewart and Walker, 1989) with slow shaking. A cold acclimation responsive cDNA clone from alfalfa, RootCAR1 (GenBank Accession AF072932), was labeled with 32P-dCTP using random priming (Feinberg and Vogelstein, 1983). Hybridization and washing of membranes were done as described by Gana et al., (1997). Membranes were exposed to x-ray film at -80°C.
Statistical Analysis
The experiment was replicated four times in each of 2 yr. Years were treated as random effects and germplasm pools as fixed effects. Data were analyzed as a randomized complete block design with a mixed model using analysis of variance (SAS Institute, Cary, NC). Where F-tests were significant (P 
0.05 unless otherwise stated), an LSD was calculated for mean comparisons. Regression was used to examine the relationships among variables.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plant height in October (estimate of fall dormancy) of the cultivars used as controls differed in the expected manner (Fig. 2A). Averaged across years, height of Norseman averaged 8 cm, while height of Wadi Qurayat approached 50 cm, with Saranac and Lahontan intermediate between these. For each year linear regression of fall dormancy ratings of Norseman, Saranac, Mesilla-O, Lahontan, CUF 101-O, and Wadi Qurayat vs. mean fall plant height (hgt) was significant and resulted in the equations: Year 1, y = -0.6 + 4.1hgt; Year 2, y = 8.8 + 3.9hgt; r2 
0.96. These regression equations were used to predict fall dormancy ratings of the selected populations within each of the respective years, and these estimates were analyzed using analysis of variance (Fig. 2A). Selection for greater fall dormancy reduced plant heights and fall dormancy ratings for each cycle of selection, CUF 101-L1 through CUF 101-L3. A similar pattern of reduction in height and fall dormancy rating in response to selection for greater fall dormancy occurred in Mesilla, but differences between the L1 and L2 populations, and the L2 and L3 populations were not statistically significant. Selection for less fall dormancy in CUF 101 had no effect on height and fall dormancy rating of this already fall nondormant cultivar, whereas only Mesilla H3 was significantly taller and less fall dormant than Mesilla-O, -H1, and -H2 (Fig. 2A).
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0.01) for both CUF 101 and Mesilla populations. Averaged over both years, fall height of Mesilla was reduced by 2.1 cm/cycle of selection, while height of CUF 101 was reduced 5.7 cm/cycle of selection. Winter injury of the populations differed. Averaged across both years, Norseman and Saranac had less winter injury than Mesilla-O and Lahontan, both of which incurred less injury than CUF 101-O and Wadi Qurayat (Fig. 2B). Winter injury of Mesilla-L1, -L2, and -H1 populations were not statistically different from Mesilla-O, whereas injury of Mesilla-L3 was reduced and that of Mesilla-H2 and -H3 populations increased by selection. Winter injury of CUF 101-H1 to -H3 was very high and unchanged by selection for less fall dormancy when compared to CUF 101-O. Selection for greater fall dormancy reduced winter injury of all CUF 101-L populations when compared to CUF 101-O, with injury of CUF-L3 being reduced to values not statistically different from that of Saranac.
Reduction in shoot height in October (fall dormancy) and winter survival were positively associated (Fig. 3). Although the relationship differed slightly between years due to management (Table 1) and environment (Fig. 1), a positive linear relationship between shoot height in October and winter injury score was observed both years (Fig. 3). The slopes of these regressions indicate that winter injury scores would increase approximately 0.4 for each 10-cm increase in shoot height in October, a value that is equivalent to the difference in winter injury scores between Norseman and Lahontan (Fig. 2B). This latter cultivar is not adapted for use in the Midwest USA because of excessive winter injury most years.
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Protein concentrations of roots sampled in December differed among cultivars and populations. Averaged over both years roots of Norseman, Saranac, and Lahontan had higher protein concentrations than roots of Mesilla-O, CUF 101-O, and Wadi Qurayat (Fig. 6). When compared to Mesilla-O, selection for greater fall dormancy significantly increased root protein concentrations of Mesilla-L2 and -L3, while selection for less fall dormancy reduced protein concentrations in roots of Mesilla-H3. Likewise, the more fall dormant CUF 101-L2 and -L3 had higher root protein concentrations than CUF 101-O. However, selection for less fall dormancy did not significantly reduce root protein concentrations in CUF 101-H populations, probably due, in part, to the low protein concentrations already present in roots of the parent CUF 101-O. Averaged over both years, regression of winter injury scores vs. root protein concentration revealed a significant negative linear relationship between these characteristics (Fig. 7). Under the conditions of this study increasing root protein concentration in December by 5 g/kg dry wt. did reduce winter injury scores by 0.5, equivalent to the winter injury score difference between the very winter hardy Norseman and Mesilla-O, the latter of which is considered not adapted for use under Midwest growing conditions.
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Selection for greater fall dormancy resulted in roots of CUF 101-L2 and -L3 having much higher RootCAR1 transcript levels when compared to CUF 101-O and -L1 (Fig. 8). This difference in RootCAR1 transcript abundance was associated with reduced winter injury scores and improved winter hardiness (Fig. 2B). Selection for less fall dormancy in CUF 101-H1, -H2, and -H3 did not markedly change steady-state levels of RootCAR1 transcript, and likewise, did not alter winter injury scores when compared to CUF 101-O.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In order to create nondormant alfalfa cultivars that are winter hardy it would be useful to understand physiological, biochemical, and genetical mechanisms controlling fall dormancy and winter survival. In this experiment, we studied alfalfa populations derived by three cycles of disruptive selection for contrasting fall dormancy using either CUF 101 or Mesilla as parents. We observed incremental reductions in fall dormancy with each cycle of selection (Fig. 2A) and with these changes reduced winter injury (Fig. 2B). Linear regression revealed a close (r2 = 0.81–0.88) association between fall shoot growth (nondormancy) and winter injury in these populations (Fig. 3). Previous work with CUF 101-O, -L3, and -H3 also showed that CUF 101-L3 was more fall dormant, and much more winter hardy than CUF 101-O (Cunningham et al., 1998). A continuum of variation in fall dormancy in response to selection, however, was not evident in that study because only the third cycle of selection was studied, and the Mesilla populations were not included. While fall dormancy has been associated with cultivar differences in winter survival in previous studies, never before has a set of populations representing consecutive cycles of alfalfa selected exclusively for contrasting fall dormancy been available to evaluate this association. Results from this study and our previous work with these populations (Cunningham et al., 1998) suggest that decreasing alfalfa fall dormancy without increasing susceptibility to winter injury will be difficult. Recently, Brummer et al. (2000) reported little association between fall growth and winter injury in an F1 population derived from a M. sativa x M. falcata cross. Based on genetic correlations and heritabilities, they suggested that both winter hardiness and fall growth can be improved simultaneously in this population. Although possible, the consistent association between fall dormancy and winter hardiness observed in our populations in this and a previous study (Cunningham et al., 1998), suggest that it may be necessary to use different germplasms than those we have studied to simultaneously improve fall growth and winter survival.
High concentrations of root total nonstructural carbohydrates (TNC, sugar + starch) is generally believed to be essential for alfalfa winter survival (Klebesadel, 1971; Sheaffer et al., 1992). Root TNC concentrations (data not shown) mirrored trends in root starch (Fig. 4B), and like starch, were lowest in Norseman the most fall dormant, winter hardy cultivar. The nondormant populations and cultivars had root starch and TNC levels that equaled or exceeded those of Norseman indicating that starch and TNC reserve levels per se cannot be used to predict genetic differences in winter survival.
Root sugar concentrations were positively associated with fall dormancy and limited winter injury (Fig. 4A and 5). Small increases in fall dormancy were accompanied by small, but consistent increases in root sugar concentrations and reduced winter injury in both CUF 101 and Mesilla. This agrees with our previous report where root sugar concentrations of CUF 101-L3 exceeded those of CUF 101-O (Cunningham et al., 1998). Root sugar concentrations in the present study were lower than those previously reported for these populations, possibly due to year-to-year differences in the environment under which plants cold acclimated. Nevertheless, the ranking of root sugar concentrations did not vary between this and our previous (Cunningham et al., 1998) study.
Other research (Castonguay and Nadeau, 1998), including our own (Cunningham and Volenec, 1998) has shown less consistency in fall dormancy-driven changes in root sugar accumulation during cold acclimation. These studies used an assortment of cultivars that differed in fall dormancy, rather than contrasting fall dormancy populations derived from a single cultivar, so genetic background differences may have confounded the cold acclimation responses. In addition, it has been suggested that accumulation of raffinose and stachyose in crowns may be more important than sucrose accumulation in enhancing alfalfa winter survival (Castonguay et al., 1995; Castonguay and Nadeau, 1998). Work is being initiated to evaluate the composition of sugars found in overwintering organs of these contrasting populations.
Like root sugars, root protein concentrations also systematically increased in response to selection for greater fall dormancy in both CUF 101 and Mesilla (Fig. 6), and these changes were positively associated with reduced winter injury (Fig. 7). We have previously reported greater accumulation of protein in roots of fall dormant alfalfa cultivars (Cunningham and Volenec, 1998), and CUF 101-L3 when compared to CUF 101-O (Cunningham et al., 1998). Electrophoretic analysis revealed that a large proportion of this protein accumulation is vegetative storage proteins (VSPs, Cunningham and Volenec, 1996) that serve as a source of N when shoot growth is initiated in spring. Although cold acclimation-induced polypeptides have been observed in fall dormant populations in previous work (Cunningham et al., 1998), SDS-PAGE analysis did not reveal new polypeptides in root protein extracts of the fall dormant, winter hardy populations in this study. This is not surprising considering that a few hundred of the thousands of polypeptides present in alfalfa roots are resolved using this technique. Changes in the abundance of key proteins associated with greater fall dormancy and enhanced winter survival could easily escape detection.
We used Northern analysis to assay transcript abundance for a gene (RootCAR1) whose expression has been associated with cultivar differences in winter hardiness (Monroy et al., 1993; Volenec, unpublished data, 2000). The high sensitivity of this assay may permit us to detect differences in gene expression that result from selection for contrasting fall dormancy that cannot be determined using SDS-PAGE analysis of proteins. Mesilla-O roots contain moderate RootCAR1 transcript levels, and these increased slightly as fall dormancy increased and winter injury declined in Mesilla-L3. Selection for greater fall dormancy in CUF 101 markedly increased the steady-state transcript abundance of RootCAR1 in roots of CUF 101-L2 and -L3, and with it, greatly reduced winter injury. By comparison, no change in transcript abundance was observed with selection for less fall dormancy in CUF 101-H1, -H2, or -H3, and there was no change in fall dormancy or winter injury of these populations. This positive association between RootCAR1 transcript abundance and alfalfa winter survival is further verified by the high transcript abundance found in the very winter hardy Norseman, and the lack of transcript detected in roots of Wadi Qurayat. Even though we do not yet know the function of the RootCAR1 protein in planta, expression of this gene may serve as a useful marker for identifying winter hardy alfalfa plants in the fall of the seeding year. This would improve selection efficiency by eliminating evaluation at multiple sites over several years to identify winter hardy plants. Work is underway to determine the feasibility of this approach for alfalfa improvement.
Received for publication August 11, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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